Description of the karyotype of Rhagomys rufescens Thomas, 1886 (Rodentia, Sigmodontinae) from Southern Brazil Atlantic forest

Rhagomys rufescens (Rodentia: Sigmodontinae) is an endemic species of the Atlantic forest from Southern and Southeastern Brazil. Some authors consider Rhagomys as part of the tribe Thomasomyini; but its phylogenetic relationships remain unclear. Chromosomal studies on eight specimens of Rhagomys rufescens revealed a diploid number of 2n = 36 and a number of autosome arms FN = 50. GTG, CBG and Ag-NOR banding and CMA3 /DAPI staining were performed on metaphase chromosomes. Eight biarmed and nine acrocentric pairs were found in the karyotype of this species. The X and Y chromosomes were both acrocentric. Most of the autosomes and the sex chromosomes showed positive C-bands in the pericentromeric region. The X chromosome showed an additional heterochromatic block in the proximal region of the long arm. Nucleolus organizer regions (NORs) were located in the pericentromeric region of three biarmed autosomes (pairs 4, 6 and 8) and in the telomeric region of the short arm of three acrocentrics (pairs 10, 12 and 17). CMA 3 /DAPI staining produced fluorescent signals in many autosomes, especially in pairs 4, 6, and 8. This study presents cytogenetic data of Rhagomys rufescens for the first time.

The objective of this study was to describe the karyotype of Rhagomys rufescens from southern Brazil after conventional and CMA 3 /DAPI staining, and GTG, CBG and Ag-NOR banding. The chromosomal data presented in this work can provide additional information for studies on both taxonomic and phylogenetic relationships.
Eight specimens (five males and three females) were analyzed. They were captured at PNMNG, at "Mono" locality (27°02'59" S, 49°08'57" W), in Indaial city, in the state of Santa Catarina, southern Brazil. This park is now part of Parque Nacional da Serra do Itajaí (PNSI) (Figure 1). The animals were caught in Sherman traps placed at 3 m from the ground, according to Kierulff et al. (1991), with adaptations.
Skins and skulls of specimens were deposited at the Coleção Zoológica da Fundação Universidade Regional de Blumenau (CZFURB), in Blumenau, State of Santa Catarina, Brazil.
Analyses after conventional staining showed 2n = 36 and FN = 50 in all specimens (Figure 2a), with five metacentric pairs (1, 3, 6 and 8), three submetacentric (pairs 2, 4 and 5) and nine acrocentric pairs (pairs 9 to 17), decreasing gradually in size. The X chromosomes were acrocentric, indistinguishable from pair 9, whereas the Y chromosome was also acrocentric and similar in size to pair 10. All chromosome pairs, including the sex chromosomes, could be identified after G-banding. The X chromosome showed two positive bands in the medium portion of the long arm and the Y chromosome had one proximal band in the long arm ( Figure 2b). C-banding revealed pericentromeric constitutive heterochromatic blocks in most autosomes and also in the sex chromosomes. An additional interstitial C-band was present in the proximal region of the long arm of the X chromosome ( Figure 2c).
NORs were detected in the pericentromeric region of pairs 4, 6 and 8, and in the telomeric region of the short arm of acrocentric pairs 10, 12 and 17 ( Figure 2d). Two to twelve NORs were observed, with a mean of 7.33 ± 3.19 per cell (N = 39). 480 Testoni et al.   The double staining with the GC-and AT-specific fluorochromes, CMA 3 and DAPI, respectively, showed intense fluorescent CMA 3 signals in the pericentromeric region of pairs 4 and 6, and throughout the length of pair 8. Less intense signals were observed in other pairs (Figure 2e).
Rhagomys is a polytypic genus composed by R. longilingua and R. rufescens. After comparative morphological analyses, Pacheco (2003) considered it monophyletic, although the two forms show a discontinuous distribution: R. longilingua can be found in Peru and Bolivia, whereas R. rufescens occurs in southern and southeastern Brazil. The phylogenetic relationships of this genus with other Sigmodontinae are controversial and uncertain and it has been previously included in different tribes of this subfamily.
After analyses of the nuclear IRBP (Interphotoreceptor Retinoid Binding Protein) gene sequences, D'Elia et al. (2006) suggested grouping Rhagomys longilingua with the Thomasomyini species as a sister-group of Thomasomys and as part of a larger clade that also includes Aepeomys and Rhipidomys.
On the other hand, Percequillo et al. (2004), based on mitochondrial cytochrome B sequences, concluded that the position of R. rufescens within Sigmodontinae was uncertain and that Rhagomys was either closely associated to Andinomys, followed by a Thomasomys-Rhipidomys group, or closer to Juliomys, followed by Andinomys.
The cytogenetic data of Rhagomys rufescens (2n = 36 and FN = 50) described herein are the first for this genus. Pair 9 and the X chromosome were undistinguishable after conventional staining because of their similar sizes and morphologies. However, GTG and CBG banding patterns showed significant differences allowing their individual identification. The two interstitial G-bands in the long arm of the X chromosome, characteristic of mammalian X chromosomes (Pathack and Stock, 1974), could be observed. Furthermore, an additional block of interstitial constitutive heterochromatin was present in the proximal region of the long arm of X chromosome, whereas pair 9 only presented a pericentromeric heterochromatic block. The Y chromosome, which is almost completely heterochromatic in many species of South-American rodents (Sbalqueiro et al. 1991;Andrades-Miranda et al., 2001), only presented a positive C-band in the pericentromeric region in Rhagomys rufescens.
After double fluorochrome staining, CMA3-positive and DAPI-negative signals were present in sites coincident with all AgNORs. The correlation of NORs with GC-rich sites is relatively common among vertebrates (Schmid, 1982;Amemiya and Gold, 1986;Artoni et al., 1999, among others), although the reverse correlation is not always valid. Additional GC-rich sites were also observed, mainly in the first three chromosome pairs. These sites were euchromatic domains adjacent to G-bands, known to correspond to GCrich isochores (R-bands), especially close to the telomeric region (Bernardi, 1993;Holmquist and Ashley, 2006). However, several authors suggested the use of the silver staining technique in conjunction with FISH (rDNA probes) to confirm the number and location of NORs (Santos et al., 2001;Fagundes et al., 2003;Leite-Silva et al., 2003).
The comparison of the chromosome data presented herein to those of the other Thomasomyini species mentioned above does not allow to determine the taxonomic relationship of Rhagomys rufescens. The scarcity of cytogenetic data of a larger number of species and the lack of techniques that could show more details about chromosome structure makes further taxonomic analysis a difficult task. It is evident that several chromosome rearrangements have contributed to the karyotypic variability observed in Thomasomyini. Complementary data obtained from differential staining associated with FISH techniques, such as ZOO-FISH (Hass et al., 2008), is necessary for clarifying the mechanisms of karyotypic evolution in this group, and hence contribute to determine the taxonomic position of this genus. The data reported herein are important as a first characterization of the chromosome complement of R. rufescens because it allows the identification of some primary features of its karyotype.